De Novo Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Chaenomeles speciosa (Sweet) Nakai Revealed the Existence of Two Structural Isomers

As a valuable Chinese traditional medicinal species, Chaenomeles speciosa (Sweet) Nakai (C. speciosa) is a natural resource with significant economic and ornamental value. However, its genetic information is not well understood. In this study, the complete mitochondrial genome of C. speciosa was assembled and characterized to explore the repeat sequences, recombination events, rearrangements, and IGT, to predict RNA editing sites, and to clarify the phylogenetic and evolutionary relationship. The C. speciosa mitochondrial genome was found to have two circular chromosomes as its major conformation, with a total length of 436,464 bp and 45.2% GC content. The mitochondrial genome contained 54 genes, including 33 unique protein-coding genes, 18 tRNAs, and 3 rRNA genes. Seven pairs of repeat sequences involving recombination events were analyzed. Both the repeat pairs, R1 and R2, played significant roles in mediating the major and minor conformations. In total, 18 MTPTs were identified, 6 of which were complete tRNA genes. There were 454 RNA editing sites in the 33 protein-coding sequences predicted by the PREPACT3 program. A phylogenetic analysis based on 22 species of mitochondrial genomes was constructed and indicated highly conserved PCG sequences. Synteny analyses showed extensive genomic rearrangements in the mitochondrial genome of C. speciosa and closely related species. This work is the first to report the C. speciosa mitochondrial genome, which is of great significance for conducting additional genetic studies on this organism.


Introduction
C. speciosa, belonging to the genus Chaenomeles in the Rosaceae family, is a native temperate plant widely cultivated in Asia and Europe. C. speciosa has been widely used in medicine and the functional food industries [1,2]. The dried fruit of C. speciosa has been utilized in traditional Chinese medicine for thousands of years. Previous studies have shown that the chemical components of C. speciosa extracts are rich in flavonoids, phenolics, terpenoids, and phenylpropionic acids [3][4][5]. The People's Republic of China's Pharmacopoeia lists that oleanolic acid and ursolic acid are two triterpenoid acids that can be used in medicine. Several traditional C. speciosa cultivars have recently been released on the market and undergone increased production [6].
Mitochondria are involved in numerous metabolic processes and perform crucial roles in cell differentiation, apoptosis, cell development, and cell division by converting biomass energy into chemical energy for daily activities [7][8][9]. On the other hand, the mitochondrion is a semi-autonomous organelle with a mitochondrial genome genetic system distinct from the cell nucleus and typically exhibits maternal inheritance [10,11]. Coordinated nucleocytoplasmic interaction is essential for plant growth. Artificial hybridization between or within plant species could change the balance of cytonuclear interactions, often used to breed new varieties [12]. Cytoplasmic male sterility (CMS), driven by a genetic conflict between the nuclear and mitochondrial genomes, is a typical example and has been reported in many species [13].
Plant mitochondria contain the largest and most complex organelle genomes, with various sizes and structures [14,15]. These mitochondria are recognized as obtaining foreign sequences through intracellular gene transfer (IGT) and horizontal gene transfer (HGT) [16][17][18][19]. Numerous well-known genes, introns, and varied intergenic regions can be found in mitochondrial genomes [20]. The size of mitochondrial genomes varies significantly, by up to three orders of magnitude, because of differences in non-coding regions, such as repeat sequences, introns, intracellularly transferred sequences from plastids (mitochondrial plastid sequences, MTPTs), and horizontal gene transfer from foreign donors [21]. As previously reported, the Viscum scurruloideum mitochondrial genome exhibited a size of 66 Kb [22], while the Lactuca sativa and Siberian larch mitochondrial genomes were estimated at 11.7 Mb [23,24]. The mitochondrial genome was previously thought to have a conventional, single-circular chromosome structure. However, previous reports indicate that the conformations are diverse, with polycyclic being the most common in Cucumis sativus [25] and a variety of linear, circular, and branched structures in Quercus acutissima [26]. Moreover, the multi-chromosomal mitochondrial genome has been reported in many species. For example, 54 distinct circular chromosomes in Lophophytum mirabile range from 7.2 to 580 kb in size [27], while 21 smaller circular chromosomes in Rhopalocnemis phalloides range from 4.95 to 7.86 kb [28].
Plant mitochondrial genomes have a dynamic structure with multiple configurations [29,30]. It has generally been accepted that large repeats of several kilobases of DNA mediate the presence of multiple mitochondrial genome conformations via recombination at a high frequency [31]. Emily et al. [32] found that only 13 of the 72 species of angiosperms showed no repeats with lengths exceeding 600 bp, and the repeats were more extensive and more frequent in vascular plants. Further, the accumulation of sub-genomic interconvertible through recombination events could change the flexibility of plant mtDNA and help mtDNA evolve through genetic diversity [14,31,33]. The abundance of repeat sequences is typically related to mitochondrial genome structural rearrangements [34]. In addition to the mitochondrial genome's variability in genome size, gene content, and genomic structure, functional genes also differ significantly due to post-transcriptional editing, namely RNA editing, which may lead to incredibly diverse gene sequences [35,36]. Despite the high rate of rearrangement and recombination, the mitochondrial genome typically shows a low mutation rate, which may result from the mitochondrial repair processes [14,33,37].
Understanding genetic information is of great significance, but, until now, only the chloroplast genome of C. speciosa has been sequenced and studied [38]. In this study, the mitochondrial genome of C. speciosa was first assembled and annotated. This study aimed to analyze the mitochondrial genome structure mediated by repeated sequences, IGTs, RNA editing sites, and selective pressure and explore the synteny and phylogenetic relationships. Our results expand the genetic information in Rosaceae and provide a theoretical basis for further utilization and genomic breeding studies of the C. speciosa species.

Assembly and Annotation of the Mitochondrial Genome and Chloroplast Genome
The organelle genome in this study was assembled using a hybrid assembly strategy. First, GetOrganelle (version v1.7.5) [42] was used to obtain short reads of the chloroplast and mitochondrial genomes separately. The short reads were then assembled by SPADES [43] into a unitig graph. The contigs in the unitig graph served as the reference genomes for the nanopore read mapping using BWA software [44]. The mapped reads were extracted using Samtools (v1.9) [45], and then the long reads mapped to the repeat region of the unitig graph were used for solving the repeat region. Finally, the contigs were merged using bandage software [46] to form the circular DNA molecule. The mitochondrial genome was annotated by the Geseq software (v2.03) [47]. CPGAVAS2 (v1.0) [48] was utilized to annotate the chloroplast genome with Database 3. The chloroplast genome annotation was checked via the CPGView web server (v1.0) [49].

Analysis of Repeated Sequences and Genome Recombination
BLASTN [50] was used to detect the repeated sequences in the C. speciosa mitochondrial genome, followed by manual exclusion. Then, two repeated units of the repeated sequences and their flanking 1000 bp regions were extracted as the primary conformation. Afterward, the repeat unit's 1000 bp regions upstream and downstream were exchanged to simulate the secondary conformation that could result from artificial recombination. BWA software mapped the nanopore data to major and minor conformation sequences [44]. By counting the number of long reads that completely overlapped the regions of the repeated sequence, it was feasible to determine whether there was mitochondrial genome recombination.

DNA Transfer and Phylogenetic Tree Construction
Due to the lack of a published nuclear genome for C. speciosa, only two organelle genomes can be utilized to analyze intracellular sequence migration. To identify the MTPTs, the chloroplast genome of C. speciosa was compared with the mitochondrial genome using the BLASTN program with the parameter "-evalue 1e-5". The results were visualized using TBtools (v1.112) [51].

RNA Editing Site Identification and Synteny Analysis
The PCGs of C. speciosa were extracted using PhyloSuite software. The extracted PCGs were submitted to the PREPACT3 web server (http://www.prepact.de/prepact-main.php, accessed on 3 August 2022) for RNA editing site prediction with the default parameters [58].
The mitochondrial genome data of seven species (Pyrus betulifolia, Sorbus torminalis, Malus domestica, Eriobotrya japonica, Prunus avium, Rosa chinensis, and Fragaria orientalis) from the Rosaceae family that properly represent their genera were downloaded from the NCBI public database at https://www.ncbi.nlm.nih.gov (accessed on 3 August 2022). BLASTN was performed to compare eight mitochondrial genomes pairwise and obtain homologous sequences. For the Multiple Synteny Plot, conserved colinear blocks longer than 0.5 Kb were selected. The Multiple Synteny Plot of C. speciosa with the seven species was constructed based on sequence similarity using MCscanX [59].

General Features of the C. speciosa Mitochondrial Genome
The major conformation of the C. speciosa mitochondrial genome was two circular chromosomes, with a total length of 436,464 bp and 45.2% GC content ( Figure 1). The lengths of the two circular chromosomes (chromosome 1 and chromosome 2) were 307,720 bp and 128,744 bp, and the GC contents were 45.15% and 45.33%, respectively (Table 1).
BLASTN was performed to compare eight mitochondrial genomes pairwise and obtain homologous sequences. For the Multiple Synteny Plot, conserved colinear blocks longer than 0.5 Kb were selected. The Multiple Synteny Plot of C. speciosa with the seven species was constructed based on sequence similarity using MCscanX [59].

Repeats and Homologous Recombination
As shown in Figure 2, seven pairs of repeated sequences were detected as being involved in mediating mitochondrial genome recombination, including R1 and R2 as long repeats (>1000 bp), R15 as short repeats (<100 bp), and R4, R5, R7, and R14 as medium-sized repeats. Among them, R4, R7, R14, and R15 were reverse repeats; R1, R2, and R5 were forward repeats. In addition, R2, R7, and R14 were located separately in chromosomes 1 and 2, and the other four pairs of repeated sequences were located in chromosome 1 ( Table 3).
After simplifying, four chromosomes were presented (Table 4) and mediated by R1 and R2. As shown in Figure 3, R1 and R2 may mediate mtDNA formation into three independent circular chromosomes or one united circular chromosome. The major conformation was two circular chromosomes, which were supported by the majority of long reads. R1, with the most extensive length, was 7887 bp and obtained similar rates, supporting long reads for both routes from the major conformation (combined in one circular) and minor conformation (divided into two circulars) (21 + 20/16 + 18). In contrast, another pair of R2 repeated sequences allowed for many more reads on a single circular chromosome than on two circular chromosomes (168 + 100/3 + 0). Genes 2023, 14, x FOR PEER REVIEW 6 of 17  After simplifying, four chromosomes were presented (Table 4) and mediated by R1 and R2. As shown in Figure 3, R1 and R2 may mediate mtDNA formation into three independent circular chromosomes or one united circular chromosome. The major conformation was two circular chromosomes, which were supported by the majority of long reads. R1, with the most extensive length, was 7887 bp and obtained similar rates, supporting long reads for both routes from the major conformation (combined in one circular) and minor conformation (divided into two circulars) (21 + 20/16 + 18). In contrast, another pair of R2 repeated sequences allowed for many more reads on a single circular chromosome than on two circular chromosomes (168 + 100/3 + 0).   Table 4. The segments and repeats are contained in four chromosomes. In addition, as large repeats involving recombination events, R1 exhibited a recombination frequency of 45.33%, while R2 showed a recombination frequency of 1.1%. The other repeats were involved in recombination, with frequencies ranging from 0.22% to 2.27%.

Chromosome
Order in Contigs In addition, as large repeats involving recombination events, R1 exhibited a recombination frequency of 45.33%, while R2 showed a recombination frequency of 1.1%. The other repeats were involved in recombination, with frequencies ranging from 0.22% to 2.27%.

Gene Transfers between Chloroplast and Mitochondrial Genomes
According to the sequence similarity analysis, 18 MTPTs (Table S1), with a total length of 3237 bp, accounted for 0.74% of the total length of the mitochondrial genome ( Figure 4). Among them, five MTPTs exceeded a length of 100 bp, with MTPT11 and MTPT12 being the longest at 879 bp. By annotating these homologous sequences, six complete tRNA genes were identified, including trnD-GUC, trnH-GUG, trnI-CAU, trnM-CAU, trnN-GUU, and trnW-CCA. In addition to two partial tRNA genes (trnA-UGC and trnA-UGC) and two partial rRNA genes, five unique partial PCGs were identified, including psbC, psbA, ndhC, atpA, and psbE.

Gene Transfers between Chloroplast and Mitochondrial Genomes
According to the sequence similarity analysis, 18 MTPTs (Table S1), with a total length of 3237 bp, accounted for 0.74% of the total length of the mitochondrial genome ( Figure 4). Among them, five MTPTs exceeded a length of 100 bp, with MTPT11 and MTPT12 being the longest at 879 bp. By annotating these homologous sequences, six complete tRNA genes were identified, including trnD-GUC, trnH-GUG, trnI-CAU, trnM-CAU, trnN-GUU, and trnW-CCA. In addition to two partial tRNA genes (trnA-UGC and trnA-UGC) and two partial rRNA genes, five unique partial PCGs were identified, including psbC, psbA, ndhC, atpA, and psbE.   Table S1 presents the detailed data for 18 MTPTs.

Phylogenetic Analysis
To understand the evolutionary status of the C. speciosa mitochondrial genome, a phylogenetic tree of 22 species from six families of angiosperm was constructed ( Figure  5). Two mitochondrial PCGs from Brassicaceae (A. alpina and B. oleracea) were established  Table S1 presents the detailed data for 18 MTPTs.

Phylogenetic Analysis
To understand the evolutionary status of the C. speciosa mitochondrial genome, a phylogenetic tree of 22 species from six families of angiosperm was constructed ( Figure 5). Two mitochondrial PCGs from Brassicaceae (A. alpina and B. oleracea) were established as an outgroup. The taxa from six families (Rosaceae, Rhamnaceae, Cannabaceae, Moraceae, and Ulmaceae) were well clustered. In the cluster of the Rosaceae family, species from the Pyrus, Malus, and Prunus genera were well grouped.  Table S1 presents the detailed data for 18 MTPTs.

Phylogenetic Analysis
To understand the evolutionary status of the C. speciosa mitochondrial genome, a phylogenetic tree of 22 species from six families of angiosperm was constructed ( Figure  5). Two mitochondrial PCGs from Brassicaceae (A. alpina and B. oleracea) were established as an outgroup. The taxa from six families (Rosaceae, Rhamnaceae, Cannabaceae, Moraceae, and Ulmaceae) were well clustered. In the cluster of the Rosaceae family, species from the Pyrus, Malus, and Prunus genera were well grouped.

RNA Editing Sites Prediction
Four hundred fifty-four potential RNA editing sites were predicted on 33 mitochondrial PCGs (Table S2). The predicted RNA editing sites in each gene are shown in Figure 6. All the RNA editing events ranged from C to U. The ccmFN and the mttB genes were edited most frequently, 38 and 34 times, respectively. In total, eight genes (atp9, rps1, rps12, sdh4, atp8, rps3, rpl10, and nad4L) were edited no more than five times. No potential RNA editing events were predicted in the nad4L gene.

Synteny Analysis
The colinear blocks were not arranged in the same order among individual mitochondrial genomes (Figure 7). A significant number of blocks was detected among C. speciosa, M. domestica, and E. japonica. R. chinensis, S. torminalis, and P. betulifolia shared numerous conserved colinear blocks. On the contrary, colinear blocks among R. chinensis, F. orientalis, and P. betulifolia were relatively weak.

RNA Editing Sites Prediction
Four hundred fifty-four potential RNA editing sites were predicted on 33 mitochondrial PCGs (Table S2). The predicted RNA editing sites in each gene are shown in Figure 6. All the RNA editing events ranged from C to U. The ccmFN and the mttB genes were edited most frequently, 38 and 34 times, respectively. In total, eight genes (atp9, rps1,  rps12, sdh4, atp8, rps3, rpl10, and nad4L) were edited no more than five times. No potential RNA editing events were predicted in the nad4L gene. Figure 6. Characteristics of the RNA editing sites identified in PCGs of the C. speciosa mitochondrial genome. The names of the protein-coding genes are displayed on the X-axis, and the number of RNA edits of the protein-coding genes is displayed on the Y-axis.

Synteny Analysis
The colinear blocks were not arranged in the same order among individual mitochondrial genomes (Figure 7). A significant number of blocks was detected among C. speciosa, M. domestica, and E. japonica. R. chinensis, S. torminalis, and P. betulifolia shared numerous conserved colinear blocks. On the contrary, colinear blocks among R. chinensis, F. orientalis, and P. betulifolia were relatively weak.

Evolutionary Selection Pressure Analysis
As shown in Figure 8, the dN/dS values at the species level by series-connecting 28 PCGs were conducted ( Figure 8A). In C. speciosa, the dN/dS values were 0.32-1.12, which were higher in genera Pyrus, Malus, and Sorbus than in Rosa, Fragaria, Prunus, and Eriobotrya.

Evolutionary Selection Pressure Analysis
As shown in Figure 8, the dN/dS values at the species level by series-connecting 28 PCGs were conducted ( Figure 8A). In C. speciosa, the dN/dS values were 0.32-1.12, which were higher in genera Pyrus, Malus, and Sorbus than in Rosa, Fragaria, Prunus, and Eriobotrya. Genes 2023, 14, x FOR PEER REVIEW 11 of 17 The pairwise dN/dS values of all PCGs are shown in Figure 8B. The dN/dS values of PCGs from R. chinensis, F. orientalis, P. salicina, and P. avium differed significantly. The dN/dS values of the pairwise comparison from the other eight species (E. japonica, P. betulifolia, P. pyrifolia, M. domestica, M. hupehensis, S. aucuparia, S. torminalis, and C. speciosa) varied slightly, except for three genes (cox2, matR, and rps3) from C. speciosa.
As shown in Figure 8C, the rps1 gene showed a dN/dS value over 1.0, implying possible positive selection. In comparison, the dN/dS values of the rest of the genes revealed diverse differences among species. Nad4L exhibited higher dN/dS values among R. chinensis and F. orientalis but low dN/dS values among the other eight species. Atp9, cox1, cox3, nad5, and nad9 showed low dN/dS values among all the species, which indicated they had undergone negative selection during evolution.

Features of the C. speciosa Mitochondrial Genome and IGT Events
Our study produced the first detailed characterization of the complete mitochondrial genome of C. speciosa. The size of the C. speciosa mitochondrial genome is equivalent to that of E. japonica and matches within the range of previously reported mitochondrial genomes in the Rosaceae family. The GC content of the C. speciosa mitochondrial genome is 45.20%, equaling the average 45% of the 38 species in the Rosaceae family [63].
Structurally, the two circular chromosomes forming the major conformation of the C. speciosa mitochondrial genome demonstrated multi-chromosomes, as previously reported for many plants. The mitochondrial genomes of C. sativus and Q. acutissima are assembled into three chromosomes [25]. Additionally, the mitochondrial genome of Silene conica is  Figure 8B. The dN/dS values of PCGs from R. chinensis, F. orientalis, P. salicina, and P. avium differed significantly. The dN/dS values of the pairwise comparison from the other eight species (E. japonica, P. betulifolia, P. pyrifolia, M. domestica, M. hupehensis, S. aucuparia, S. torminalis, and C. speciosa) varied slightly, except for three genes (cox2, matR, and rps3) from C. speciosa.
As shown in Figure 8C, the rps1 gene showed a dN/dS value over 1.0, implying possible positive selection. In comparison, the dN/dS values of the rest of the genes revealed diverse differences among species. Nad4L exhibited higher dN/dS values among R. chinensis and F. orientalis but low dN/dS values among the other eight species. Atp9, cox1, cox3, nad5, and nad9 showed low dN/dS values among all the species, which indicated they had undergone negative selection during evolution.

Features of the C. speciosa Mitochondrial Genome and IGT Events
Our study produced the first detailed characterization of the complete mitochondrial genome of C. speciosa. The size of the C. speciosa mitochondrial genome is equivalent to that of E. japonica and matches within the range of previously reported mitochondrial genomes in the Rosaceae family. The GC content of the C. speciosa mitochondrial genome is 45.20%, equaling the average 45% of the 38 species in the Rosaceae family [63].
Structurally, the two circular chromosomes forming the major conformation of the C. speciosa mitochondrial genome demonstrated multi-chromosomes, as previously reported for many plants. The mitochondrial genomes of C. sativus and Q. acutissima are assembled into three chromosomes [25]. Additionally, the mitochondrial genome of Silene conica is arranged into numerous circular chromosomes, though some exhibit no proteincoding capability [23].
Among the 41 protein-coding genes from the mitochondrial genome of the common ancestor of angiosperms, 33 genes were annotated in the C. speciosa mitochondrial genome, including 29 core genes and 4 non-core genes. This result indicates that the deleted relevant genes might have been transferred to the nucleus, a common phenomenon during longterm angiosperm evolution [64,65]. Transferring functional genes from the mitochondria to the nucleus is an ongoing process that has helped both the mitochondria and the nucleus change over time [12]. Liu et al. [66] found all 41 genes in Cycads and Ginkgo mitochondrial genomes. In comparison, Gnetum gnemon and Welwitschia mirabilis lost 11 genes, and 4-7 intact lost mitochondrial genes were found after further searching the transcriptomes, representing the 4-7 mitochondrial genes being transferred to the nucleus. Additionally, in Gossypium raimondii, nearly all mitochondrial genes are transferred to the nucleus on Chr1. Some of these genes have more than one copy on different chromosomes [67]. As for other genes, it is possible that they are not activated by acquiring a promoter and other regulatory elements, or they are active with low or specific undetected expression. In contrast to functional gene transfers, nonfunctional gene transfers are also possible. Mitochondrial pseudogenes have been found in the nuclei of a wide range of animal species [68].
IGTs also occurred between the chloroplast, plastid, and nucleus. In the C. speciosa mitochondrial genome, MTPTs 1-10 were located on chromosome 1, MTPTs 11-18 were located on chromosome 2, and no MTPTs were found in the repeated sequences. Six complete tRNAs were found in the mitochondrial genome that migrated from the chloroplast, which similarly occurs in other plants' mitochondria. In both Suaeda glauca [69] and G. raimondii [67], it was shown that 8 and 15 tRNAs were transferred from the chloroplast genome to the mitochondrial genome. However, protein-coding genes were less numerous and frequent.

The Conformations of C. speciosa Mitochondrial Genome Mediated by Repeated Sequences
The mitochondrial genome is likely composed of several subgenomic chromosomes produced by reversible recombination events, mediated by repeated sequences [30]. In contrast to the circular molecule previously described, the mitochondrial genome structure has appeared as a variable and dynamic combination in many species [70]. As previously reported, repeated sequences play an essential role in shaping the mitochondrial genome structures through genome rearrangements, recombination, and duplications [71]. Large repeats (>1000 bp) may induce mitochondrial genome isomerization during more frequent recombination activity, whereas short-(100 bp) and medium-sized repeated sequences (100-1000 bp) show minor to moderate recombination activity [72][73][74]. As reported in the Rosaceae family, the recombination frequency of repeated sequences ranges from 0.33% to 89.69%. In the repeats of recombination frequency over 50%, 95% are large repeats (19/20), while in the repeats of recombination frequency below 1%, short-and medium-sized repeated sequences account for 99.56% [63].
In the C. speciosa mitochondrial genome, seven pairs of repeated sequences were detected as involved in recombination events. The most extensive repeated sequences of R1 were detected, mediating the conformational changes at a frequency of nearly 50%. This finding indicates that R1 medicates the presence of the major and minor conformations in nearly equal proportions. The situation discovered in the mitochondrial genome of C. speciosa was similar to that found in the mitochondrial genome of Scutellaria tsinyunensis [73], with one extensive repeated sequence forming the major and minor conformations. Nearly equimolar recombined molecules in the mitochondrial genome were generated by large repeats [75], as found in our study, as well as those reported in Silene vulgaris [76] and Ginkgo biloba [77].
Except for R1, which has a high recombination frequency, the rest of the repeated sequences took part in recombination events at relatively low rates (less than 3% in the C. speciosa mitochondrial genome). Such low recombination frequencies have been reported in other plants. In P. salicina, nine pairs of medium-sized repeats were recombined at frequencies ranging from 0.55% to 5.7% [78]. In Nymphaea colorata, seven medium-sized repeated sequences exhibited a frequency range of 0.11% to 1.28% [72].
Short-and medium-sized repeats rarely participate in recombination, in contrast to large repeats, which have a high frequency of recombination. This can further lead to complex rearrangements [30], which have been hypothesized to involve Break-Induced Replication (BIR) [79] and Single-Strand Annealing (SSA) pathways [80]. Small repeats are generally assumed to recombine sporadically and irreversibly and produce new and stable DNA arrangements. Furthermore, recombinations of short repeat sequences may contribute significantly to heteroplasmy and the evolution of plant mtDNA [81,82]. In Arabidopsis thaliana, medium-sized repeats can recombine in response to genome damage or DNA maintenance mutants [83].

Evolutionary Analysis and dN/dS Analysis
The inconsistent order of the colinear blocks' arrangement suggested that the C. speciosa mitochondrial genome had undergone extensive genomic rearrangements, likely contributing to the evolution and diversification of the C. speciosa mitochondrial genome.
Most of the mitochondrial genes underwent neutral and negative selections and were highly conserved. The genes analyzed in the C. speciosa mitochondrial genome exhibited mostly neutral and negative selections. However, the rps1 gene, with a dN/dS value over 1.0 in most species, suggests that it might have undergone positive selection during the evolution of Rosaceae. The rps1 gene is among the most dynamic genes and has been found in rice [84], maize [85], and tobacco [86]. However, in Medicago sativa, rps1 seemed to be a pseudogene, and a functional copy was located in the nucleus [87], which might provide evidence for the intercellular transfer from the mitochondria to the nucleus.

Conclusions
In this study, the C. speciosa mitochondrial genome was assembled and annotated. The total length of the C. speciosa mitochondrial genome was 436,464 bp, with 45.2% GC content, consistent with related species from the Rosaceae family. The C. speciosa mitochondrial genome was found to be composed of multiple circular chromosomes. R1 altered the conformation of the C. speciosa mitochondrial genome by recombination events with high frequency, whereas R2 achieved it through low frequency. Extensive genomic rearrangements were exhibited in the mitochondrial genome of C. speciosa and closely related species, while the protein-coding genes were highly conserved. Our study enriches the genetic information for the genus Rosaceae and provides an essential basis for protecting genetic information and improving the molecular breeding of C. speciosa.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes14020526/s1, Table S1: The homologous DNA fragment in the mitochondrial genome of C. speciosa; Table S2: The potential RNA editing events predicted on 33 mitochondrial PCGs.
Author Contributions: P.C. and Z.X. designed the research, conducted data analysis, and wrote the manuscript. Y.H. and M.Z. collected fresh C. speciosa material and conducted the experimental work. All authors read and approved the final manuscript.